Early detection of ovarian carcinoma using P16 gene products

ABSTRACT

Increased expression of the p16 gene occurs early in the development of ovarian carcinomas. This invention detects change ovarian epithelium by measuring increases in p16 gene expression by a quantitative method that compares the levels of p16 mRNA and a control mRNA (β-tubulin) in a subject to be tested against the levels of substrates in normal subjects. A biological sample such as peritoneal fluid containing mRNA derived from a subject&#39;s ovarian epithelium is taken from the subject to be tested. The mRNA is isolated from the sample, and complementary cDNA is prepared from the isolated mRNA. Using primers to p16 target sequences and to β-tubulin control sequences, the cDNA is amplified. The resultant amplification products are quantitated as to p16 and β-tubulin gene sequences. The level p16 gene expression is assessed relative to expression levels in normal subjects. An increased level of p16 gene expression in a subject determined by this method is an indication of change in the subject&#39;s ovarian epithelium indicative of presence of a carcinoma.

RELATED APPLICATIONS

This Application claims benefit of provisional application Ser. No.:60/041,554 Filed: Mar. 21, 1996, and abandoned by operation of law as ofMar. 21, 1997.

FIELD OF THE INVENTION

This invention is in the field of molecular biology and cancerdiagnostics. More specifically, it is in the field of early detection ofcarcinomas of the ovarian epithelium.

BACKGROUND OF THE INVENTION

Ovarian cancer is the forth most common cause of death from cancer inwomen. Further, ovarian cancer is the leading cause of death among womenwith cancer of the female reproductive tract. As is the case with othercommon human carcinomas, a series of multiple genetic alterations arebelieved to be involved in the development of ovarian cancer. Somegenetic abnormalities can alter the normal function of tumor suppressorgene products quantitatively and/or qualitatively and contribute tocarcinogenesis. However, the genetic alterations involved in ovariancarcinoma remain largely unknown.

Despite the advances in imaging techniques and the availability of serumtumor markers (such as CA125), the majority of ovarian cancer patientsare still diagnosed at an advanced stage of the disease--Stage III orIV. Although surgery and intensive chemotherapy have improved to alimited extent the response of ovarian cancer patients to treatment,recurrence and mortality among these patients remains a major problem.Clearly, the development of an early indicator of risk of ovarian cancerwill be useful as a tool for early diagnosis and improving prognosis.

The p16 gene (or MST1 gene) has also been identified as a putative tumorsuppressor gene. By binding to and inhibiting cyclin-dependent kinase(CDK4), which is activated by cyclin-D in the G1 phase of the cellcycle. It plays a critical role in regulation of normal cell growth. p16could suppress cell division in a similar fashion to p21 by inhibitingthe activity of cyclin-CDK complex. In addition, the p16 gene hasalready been shown on a high frequency of mutation in tumor cell lineshowever a much lower mutation frequency was detected in primary tumors.

SUMMARY OF THE INVENTION

An object of the present invention is a method for detecting change inthe ovarian epithelium of a human subject, and especially detection ofovarian tumors and carcinomas. This may be accomplished by taking abiological sample from the subject containing a p16 gene product, suchas p16 mRNA, derived from said subject's ovarian epithelium; measuringthe p16 geneproduct, such as by isolating the p16 mRNA from the sample;preparing complementary cDNA to the mRNA; combining the prepared cDNAwith primers complementary to p16 DNA target sequences and to a controlDNA target sequences; amplifying the DNA in the sample to produceamplification products; quantitating the amplification products; andcomparing the quantity of p16 target sequence amplification product inthe subject's sample against the quantity of p16 target sequenceamplification product from a similarly treated different sample todetect a change in the subject's ovarian epithelium relative to thedifferent sample. The preferred p16 target sequences are about 274 bp to546 bp long.

The different sample may be taken from the same subject that is beingtested, from a subject known to have a normal ovarian epithelium, orfrom a subject known to have an ovarian carcinoma. The biological samplemay be peritoneal fluid or any other sample that contains p16 geneproduct. "p16 gene product" is defined as the p16 gene itself and any ofits products, or expression products, including p16 cDNA, p16 mRNA andp16 proteins.

Both internal controls (in the same reaction mixture as the sample) andexternal controls (in a separate reaction mixture from the sample) maybe practiced in this method. Preferred control are non p16 gene productsthat appear at substantially the same level in both normal and tumorsamples. β-tubulin represents one such control.

Measurement and quantitation may be accomplished by any of a number ofmeans known to one skilled in the art, including gel electrophoresis,isotopic labeling and immunoassay with radioactive or other detectionmethod.

Another object of the present invention is to provide a kit fordetecting change in the ovarian epithelium of a human subject. The kitcomprises a container, reagents for measuring the relative level of ap16 gene product in a biological sample. Preferably the kit will includeinstructions. Reagents for measuring include those for quantitating thelevel of a p16 gene product in a biological sample. Additionally, thekit may include a calibrated chart in which the response of the reagentsto known levels of a p16 gene product is plotted, and incorporates useof a control, such as a β-tubulin gene product, as a calibrationstandard.

A further object of the present invention is a method for earlydetecting an ovarian carcinoma in a human subject. This is accomplishedby taking a biological sample from said subject, the sample containing ap16 gene product derived from said subject's ovarian epithelium;isolating the p16 gene product from the sample; and measuring the p16expression. For example isolating p16 mRNA from the sample, preparingcomplementary cDNA to the mRNA; combining the prepared cDNA with primerscomplementary to p16 DNA target sequences and to one or more control DNAtarget sequences; amplifying the DNA in the sample to produceamplification products; quantitating the amplification products; andcomparing the quantity of p16 target sequence amplification product inthe subject's sample against the quantity of p16 target sequenceamplification product from a similarly treated reference sample todetect a change in the subject's ovarian epithelium relative to thereference sample. The reference sample may be taken from the groupconsisting of a sample from the same subject, a normal sample from asubject known to be ovarian tumor free, and a sample from a subjectknown to have an ovarian tumor.

Another object of the present invention is a method for producing acalibrated chart by collecting a biological sample from each member of afirst group of subjects known to bear an ovarian tumor, and from eachmember of a second group of subjects known to be free of an ovariantumor; quantitating the presence of a p16 gene product in each sample,the gene product selected from the group consisting of p16 cDNA, p16mRNA and p16 protein; and then making a chart that shows the quantity ofthe p16 gene product for the tumor-bearing versus the tumor-free groupsof subjects. The quantity of p16 target sequence amplification productin the subject's sample is compared against the calibrated chart of p16target sequence amplification product from similarly treated normalsamples to detect a change in the subject's ovarian epithelium relativeto normal subjects. Alternatively, early detection of an ovarian tumorcan be accomplished in a subject by comparing the quantity of p16 targetsequence amplification product in the subject's sample is comparedagainst the quantity of p16 target sequence amplification product fromsimilarly treated samples from subjects known to have ovarian tumors.Such early detection of ovarian tumors can be accomplished bydetermining p16 protein expression in the sample to early detect thepresence of an ovarian tumor and comparing it with the calibrated chart.

The data base for this chart will allow comparison of p16 gene productexpression levels and mutation status with other possible cancer relatedgenes in individual ovarian tumors. The relationship of gene productexpression and mutation and the effect this relationship has onmalignant potential can provide important insight into candidate geneswhich have diagnostic potential and which may be candidates for genetherapy.

While a number of objects and advantages are disclosed above, it is wellwithin the ability of one skilled in the art, in view of the presentteachings, to conceive of additional objects and advantages that arestill within the scope and spirit of the invention as a whole asdisclosed herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Linearity of β-tubulin and the p16 amplification products out to30 amplification cycles.

FIG. 2. p16 expression relative to β-tubulin in normal ovary, benignovarian tumor, LMP tumor and ovarian cancer. p16 expression issignificantly elevated in many ovarian tumor cases.

FIG. 3. p16 and β-tubulin PCR amplification products from normal ovaryand ovarian cancer. Case 4, Case 3 and Case 6 are normal ovaries. Case19, Case 26, Case 23 and Case 29 are ovarian cancers. p16 expressionrelative to β-tubulin is higher in cancer cases than in normal ovarycases.

FIG. 4. Western blot analysis of p16 protein. Lane 1 is normal ovary,lane 2 is ovarian cancer Case 27 showing p16 over expression, and lane 3is a Hela cell line as a positive control. P16 protein is stronglypositive in the ovarian carcinoma case and the Hela cell line, butnegative in the normal ovary sample.

FIG. 5. p16 PCR product prepared for direct sqencing.

FIG. 6. Cyclin D and CDK4/6 stimulate cell division by phosphorylatingretinoblastoma (Rb) protein. A transcription factor such as E2F isreleased and activates the transition from B1 to S phase of the cellcycle. p16 binds the catalytic subunit CDK4 or CDK6 and inactivates theCyclin D - CDK4/6 complex. E2F is released from Rb and also activatesthe transcription of p16.

FIG. 7. Expression levels of p53, p21 and p16 and p53 mutation status.

DETAILED DESCRIPTION OF THE INVENTION

Samples:

Fresh surgical specimens of ovarian epithelial tumors were obtained from32 subjects. They consisted of 2 benign cystadenomas, 6 cystadenomas oflow malignant potential (LMP) and 24 cystadenocarcinomas. The specifictumor types studied are shown in Table 1.

                                      TABLE I                                     __________________________________________________________________________    Expression of p53, p21, and p16 Genes and Mutation of p53 and                  p16 Genes in Normal Ovaries and Ovarian Tumors.                              Case                                                                             Lab                  mRNA Expression                                                                         Mutation                                    No.                                                                              No.                                                                              Histology          p53                                                                              p21                                                                              p16                                                                              p53   p16                                   __________________________________________________________________________    1  858                                                                              NORMAL OVARY       N  N  N  ND    ND                                      2 868 NORMAL OVARY N N N ND WT                                                3 773 NORMAL OVARY N N N ND ND                                                4 430 NORMAL OVARY N N N ND WT                                                5 456 NORMAL OVARY N N N ND ND                                                6 768 NORMAL OVARY N N N ND WT                                                7 1065 SEROUS CYSTADENOMA N N +++  ND WT                                      8 646 SEROUS CYSTADENOMA N N N ND P*                                          9 1031 SEROUS CYSTADENOMA OF LMP N N ++ ND P*                                 10 481 SEROUS CYSTADENOMA OF LMP N - - ++++ ND WT                             11 1036 MUCINOUS CYSTADENOMA OF LMP N N ++++ ND WT                            12 1101 MUCINOUS CYSTADENOMA OF LMP - - - - ++++ ND P*                        13 919 MUCINOUS CYSTADENOMA OF LMP - - - - ++++ MUTATED WT                    14 794 MUCINOUS CYSTADENOMA OF LMP N N N ND WT                                15 1242 SEROUS CYSTADENOCARCINOMA ++ - - ++ MUTATED WT                        16 475 SEROUS CYSTADENOCARCINOMA N - - ++++ ND WT                             17 1035 SEROUS CYSTADENOCARCINOMA ++ N N WT WT                                18 1240 SEROUS CYSTADENOCARCINOMA ++ N ++++ WT WT                             19 643 SEROUS CYSTADENOCARCINOMA N N ++++ ND WT                               20 1041 SEROUS CYSTADENOCARCINOMA ++ - - ++++ ND WT                           21 1032 SEROUS CYSTADENOCARCINOMA N N N ND WT                                 22 464 SEROUS CYSTADENOCARCINOMA N - - ++++ ND WT                             23 1039 SEROUS CYSTADENOCARCINOMA N N ++++ ND WT                              24 468 SEROUS CYSTADENOCARCINOMA N - - ++++ ND P*                             25 791 SEROUS CYSTADENOCARCINOMA N N ++++ ND WT                               26 515 SEROUS CYSTADENOCARCINOMA - - - - ++++ MUTATED WT                      27 1245 SEROUS CYSTADENOCARCINOMA N N ++++ ND WT                              28 1026 SEROUS CYSTADENOCARCINOMA N N ++++ ND WT                              29 1033 SEROUS CYSTADENOCARCINOMA ++ - - ++++ MUTATED WT                      30 465 SEROUS CYSTADENOCARCINOMA N N ++++ ND WT                               31 482 SEROUS CYSTADENOCARCINOMA - - ++ ++++ WT WT                            32 1243 MUCINOUS CYSTADENOCARCINOMA  N ++++ ND WT                             33 484 MUCINOUS CYSTADENOCARCINOMA  - - ++++ MUTATED WT                       34 1244 MUCINOUS CYSTADENOCARCINOMA N - - ++++ ND WT                          35 1246 MUCINOUS CYSTADENOCARCINOMA ++ N ++++ WT WT                           36 480 ENDOMETRIOID ADENOCARCINOMA - - - - ++++ MUTATED WT                    37 474 CLEAR CELL CARCINOMA N N ++++ ND WT                                    38 473 CLEAR CELL CARCINOMA N N ++++ ND WT                                  __________________________________________________________________________     Normal range is equal to Mean ± 2SD                                        ++ Positive is equal to Mean + 2SD to + 4SD                                   - - Negative is equal to Mean - 2SD to - 4SD                                  ++++ Positive is equal to Mean + 4SD or greater                               ND, not done                                                                  WT, wild type                                                                 *, Known polymorphism                                                    

Clinical staging was determined according to the criteria of theInternational Federation of Gynecology and Obstetrics. Normal ovarieswere obtained from 6 patients who underwent surgery for benigngynecological disease.

mRNA Isolation:

mRNA was prepared from the samples using a RIBOSEP™ mRNA isolation kit(Becton Dickson Laboratories). The amount of mRNA recovered wasquantitated by UV spectrophotometry.

cDNA Synthesis:

Complementary DNA was synthesized from 1.0 ug of mRNA by random hexamerpriming using a 1ST STRAND™ cDNA synthesis kit (CLONTECH). Theefficiency of the cDNA synthesis was estimated by using CLONTECH'spositive control amplimer (G3PDH). The target sequences were amplifiedin parallel with the B-tubulin genes as an internal control. Primersused for amplification were designed to yield different sized productsbetween 274 bp-546 bp. For quantitation, 2-5 μCi of ³² PdCTP was addedto the amplification reaction mixture. Amplification products wereseparated on 2% agarose gels and the radioactivity of each product wasdetermined using PHOSPHO IMAGER™ (Molecular Dynamics). Amplificationproducts for sequencing were also prepared and direct sequencing wasperformed on them.

Quantitative Amplification:

Amplification was accomplished using Polymerase Chain Reaction (PCR)chemistry in a Thermal Cycler (Perkin-Elmer Cetus) with the reactionmixture consisting of cDNA derived from 50 ηg of mRNA, 5 ρmol of senseand antisense primers for both the target gene and the β-tubulin gene,200 μmol of dNTPs, 2-5 μCI of α-³² PdCTP and 0.25 units of Taq DNApolymerase in reaction buffer (Promega in a final volume of 25 μl. Theprimer sequences are listed in Table 2. The target sequences wereamplified in parallel with the β-tubulin gene as an internal control.Each cycle of amplification included 30 seconds of denaturation at 95degrees, 1 minute of primer annealing at 62 degrees and 1 minute ofextension at 72 degrees. Thirty cycles of PCR were performed and theproducts were separated on 2% agarose gel. The radioactivity of eachband were determined by PHOSPHO IMAGER™ (Molecular Dynamics). Initialstudies showed linearity of incorporation for B-tubulin and the targetgenes over 30 cycles.

                                      TABLE 2                                     __________________________________________________________________________    Sequences of amplification Primers                                              Used for Quantitative PCR                                                   GENE                    PRIMER                                                __________________________________________________________________________    p53   4A                                                                              (SEQ. ID. NO. 1)                                                                        SENSE 5-AGGCGCTGCCCCCACCA-3                                    4B (SEQ. ID. NO. 2) ANTISENSE 5-TTCCGTCCCAGTAGATT-3                          p21 1A (SEQ. ID. NO. 3) SENSE 5-GCCGAAGTCAGTTCCTT-3                            2B (SEQ. ID. NO. 4) ANTISENSE 5-TCATGCTGGTCTGCCGC-3                          p16 4A (SEQ. ID. NO. 5) SENSE 5-CCCCACTACCGTAAATG-3                            4B (SEQ. ID. NO. 6) ANTISENSE 5-GAGCTTTGGTTCTGCCA-3                          β-TUBULIN  (SEQ. ID. NO. 7) SENSE 5-TGCATTGACAACGAGGC-3                    (SEQ. ID. NO. 8) ANTISENSE 5-CTGTCTTGACATTGTTG-3                          __________________________________________________________________________     Each sense primers have Hind3 linker sequences (GCAAGCTT) and each            antisense primers have Kpnl linker sequences (GCGGTACC) on 5 primer ends.

Direct Sequencing:

PCR amplification was carried out using the primers listed in Table 3.Amplification products were purified using WIZARD™ PCR Preps DNAPurification System (Promega). The sequencing reaction was carried outusing a PRISM™ Ready Reaction DyeDeoxy Terminator Cycle Sequencing Kit(Applied Biosystems). To remove the excess DyeDeoxy terminators, thecompleted sequencing reaction was purified using a CENTRI-SEP™ spincolumn (Princeton Separation). An Applied Biosystems Model 373A DNASequencing System was used for direct sequence determination.

                                      TABLE 3                                     __________________________________________________________________________    Sequences of Amplification Primers                                              Used for Direct Sequencing                                                  GENE                  PRIMER                                                  __________________________________________________________________________    p53                                                                              3A                                                                              (SEQ. ID. NO. 9)                                                                         SENSE 5-CTGGCCCCTGTCATCTT-3                                      3B (SEQ. ID. NO. 10) ANTISENSE 5-TATCTGACAGCGCTCA-3                           4A (SEQ. ID. NO. 11) SENSE 5-AGGCGCTGCCCCCACCA-3                              4B (SEQ. ID. NO. 12) ANTISENSE 5-TTCCGTCCCAGTAGATT-3                          5A (SEQ. ID. NO. 13) SENSE 5-TGGAAGACTCCAGTGGT-3                              5B (SEQ. ID. NO. 14) ANTISENSE 5-CTTGAGTTCCAAGGCCT-3                         p16 1A (SEQ. ID. NO. 15) SENSE 5-CGCACCGAATAGTTACG-3                           1B (SEQ. ID. NO. 16) ANTISENSE 5-CCAGCGTGTCCAGGAAG-3                          2A (SEQ. ID. NO. 17) SENSE 5-CTTCCTGGACACGCTGG-3                              2B (SEQ. ID. NO. 18) ANTISENSE 5-CTGTAGGACCTTCGGTG-3                       __________________________________________________________________________

Each sense primers have Hind3 linker sequences (GCAAGCTT) and eachantisense primers have Kpn1 linker sequences (GCGGTACC) on 5 primerends.

p53 sequence covers from aa 90 to aa 351 (sequence goes from the middleof exon 4 to the middle of exon 10).

p16 sequence covers from aa 40 to terminal amino acid (sequence goesfrom middle of exon 1 through the end of exon 3).

Results:

mRNA expression levels of P53, p21, and p16 genes relative to B-tubulingene are shown in Table 1, FIG. 1, FIG. 2 and FIG. 3. Cases withoverexpression p16 are shown in Table 4. The expression levels of p53relative to B-tubulin were higher in 7 ovarian cancer cases and lower in4 cases. p53 levels were also lower in 2 low malignant potential tumors.The expression levels of p53 relative to β-tubulin were higher in someovarian cancer cases compared to normal ovaries. p21 expression levelsof tumor were often lower than that of normal ovaries. In contrast tothe fluctuation of results observed with p53 expression, p16 expressionis more consistently significantly elevated in many ovarian cancer casesincluding some cases where p53 is mutated. Some tumors showed elevatedp53 expression, low p21 expression and high p16 expression.

                  TABLE IV                                                        ______________________________________                                        Overexpression of p16 in Ovarian Carcinoma                                                            p16 + 2SD                                               N or greater                                                                ______________________________________                                        Normal ovary     6       0 (0%)                                                 Benign tumor 2  1 (50%)                                                       LMP 6  5 (83%)                                                                Carcinoma Stage 24  32 (92%)                                                  stage 1/2 4  3 (75%)                                                          stage 3/4 20  19 (95%)                                                        Type                                                                          serous 17  15 (88%)                                                           mucinous 4  4 (100%)                                                          endomtrioid 1  1 (100%)                                                       clear cell 2  2 (100%)                                                      ______________________________________                                    

p21 expression levels were lower in 3 LMP tumors and in 10 ovariancancer cases than that of normal ovaries. One ovarian cancer case showedhigher p21 expression (Case 31). p16 expression was significantlyelevated in 28 of 32 tumors studied. Normal levels of p16 were found inonly one serous adenoma, one mucinous adenoma of LMP and 2 serouscarcinomas.

Out of the 13 ovarian tumor cases with over expression or underexpression of p53, six p53 mutated cases were found and all 6 casesresulted in amino acid changes (Table 1). According to the quantitativePCR data, 3 out of these 6 cases showed over-expression of p53 and 3cases showed under expression of p53. In addition to that, all 6 casesshowed p21 under expression (FIG. 7). One ovarian cancer (Case 33)showed one extra p53 band as well as the expected sized band (FIG. 2).The expected sized product had a T to A transversion in codon 206. Thelarger sized product had a G to A transition at the 3' splicing site ofintron 7 that resulted in intron 7 insertion. Ovarian cancer case #26showed one extra p53 product as well as the expected sized p53 product(FIG. 5). The smaller sized product had a 58 bp deletion from codon 106to codon 125 resulting in a stop codon at codon 150.

Only four p16 mutated cases were found in the 32 ovarian tumor cases weexamined (Tables 1 & 5). One benign ovarian tumor (Case 8) showed a G toT transversion in codon 127 and this transversion had been described asa polymorphism. Both one LMP tumor (Case 9) and one ovarian cancer (Case24) showed the same G to A

                                      TABLE V                                     __________________________________________________________________________    Ovarian Tumors with p16 Polymorphisms                                         Case             FIGO                                                           No. Tumor Type Stage Exon Codon Base Change Amino Acid Change               __________________________________________________________________________     8  S CYSTADENOMA    EXON2                                                                              aa127.sup.a                                                                        GCA--TCA                                                                             Ala--Ser                                   9 S CYSTADENOMA (LMP) 1 EXON2 aa148.sup.b GCG--ACG Ala--Thr                  12 M CYSTADENOMA (LMP) 1 EXON2 aa68 GCG--GCA No Change                        24 S CARCINOMA 3 EXON2 aa148.sup.b GCG--ACG Ala--Thr                        __________________________________________________________________________     .sup.a G--T transversion in codon 127 has been described as polymorphism.     .sup.b G--A transition in codon 148 has been described as polymorphism.       S; Serous                                                                     M; Mucinous                                                              

transition in codon 148 and this transition has also been described as aprevalent polymorphism. Excluding these three cases, the only p16mutation was found in an LMP tumor (Case 12) in which a G to Atransition in codon 68 was noted. This mutation however did not resultin amino acid change. These data indicate that p16 expression wassignificantly elevated in 28 out of 32 ovarian tumor cases tested. p16is seldom mutated in ovarian tumors. These data indicate that elevationin p16 expression is a direct positive indication of changes in theepithelium characteristic of ovarian carcinomas. Detection of p16 geneproducts in fluid samples is an especially sensitive means for earlydetection of changes in the ovarian epithelium.

While the above description contains many specificities, these shouldnot be construed as limitations on the scope of the invention, butrather as an exemplification of one preferred embodiment thereof. Manyother variation are possible, which would be obvious to one skilled inthe art. Accordingly, the scope of the invention should be determined bythe scope of the appended claims and their legal equivalents, and notjust by the embodiments.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                   - -  - - (1) GENERAL INFORMATION:                                             - -    (iii) NUMBER OF SEQUENCES:  18                                         - -  - - (2) INFORMATION FOR SEQ ID NO: 1:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17 BASE - #PAIRS                                                  (B) TYPE:   NUCLEIC - #ACID                                                   (C) STRANDEDNESS:  SING - #LE                                                 (D) TOPOLOGY:  LINEAR                                                - -     (ii) MOLECULE TYPE:  OTHER NUCLEIC ACI - #D                           - -    (iii) HYPOTHETICAL:  NO                                                - -     (iv) ANTI-SENSE:  NO                                                  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #1:                           - - AGGCGCTGCC CCCACCA             - #                  - #                      - #   17                                                                  - -  - - (2) INFORMATION FOR SEQ ID NO: 2:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17 BASE - #PAIRS                                                  (B) TYPE:  NUCLEIC A - #CID                                                   (C) STRANDEDNESS:  SING - #LE                                                 (D) TOPOLOGY:  LINEAR                                                - -     (ii) MOLECULE TYPE:  OTHER NUCLEIC ACI - #D                           - -    (iii) HYPOTHETICAL:  NO                                                - -     (iv) ANTI-SENSE:  YES                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #2:                           - - TTCCGTCCCA GTAGATT             - #                  - #                      - #   17                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO: 3:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17 BASE - #PAIRS                                                  (B) TYPE:  NUCLEIC A - #CID                                                   (C) STRANDEDNESS:  SING - #LE                                                 (D) TOPOLOGY:  LINEAR                                                - -     (ii) MOLECULE TYPE:  OTHER NUCLEIC ACI - #D                           - -    (iii) HYPOTHETICAL:  NO                                                - -     (iv) ANTI-SENSE:  NO                                                  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #3:                           - - CCGAAGTCA GTTCCTT             - #                  - #                      - #    17                                                                   - -  - -  - - (2) INFORMATION FOR SEQ ID NO: 4:                               - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17 BASE - #PAIRS                                                  (B) TYPE:  NUCLEIC A - #CID                                                   (C) STRANDEDNESS:  SING - #LE                                                 (D) TOPOLOGY:  LINEAR                                                - -     (ii) MOLECULE TYPE:  OTHER NUCLEIC ACI - #D                           - -    (iii) HYPOTHETICAL:  NO                                                - -     (iv) ANTI-SENSE:  YES                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #4:                           - - TCATGCTGGT CTGCCGC             - #                  - #                      - #   17                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO: 5:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17 BASE - #PAIRS                                                  (B) TYPE:  NUCLEIC A - #CID                                                   (C) STRANDEDNESS:  SING - #LE                                                 (D) TOPOLOGY:  LINEAR                                                - -     (ii) MOLECULE TYPE:  OTHER NUCLEIC ACI - #D                           - -    (iii) HYPOTHETICAL:  NO                                                - -     (iv) ANTI-SENSE:  NO                                                  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #5:                           - - CCCCACTACC GTAAATG             - #                  - #                      - #   17                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO: 6:                                    - 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#CID                                                   (C) STRANDEDNESS:  SING - #LE                                                 (D) TOPOLOGY:  LINEAR                                                - -     (ii) MOLECULE TYPE:  OTHER NUCLEIC ACI - #D                           - -    (iii) HYPOTHETICAL:  NO                                                - -     (iv) ANTI-SENSE:  YES                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #8:                           - - CTGTCTTGAC ATTGTTG             - #                  - #                      - #   17                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO: 9:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17 BASE - #PAIRS                                                  (B) TYPE:  NUCLEIC A - #CID                                                   (C) STRANDEDNESS:  SING - #LE                                                 (D) TOPOLOGY:  LINEAR                                                - -     (ii) MOLECULE TYPE:  OTHER NUCLEIC ACI - #D                           - -    (iii) HYPOTHETICAL:  NO                                                - -     (iv) ANTI-SENSE:  NO                                                  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #9:                           - - CTGGCCCCTG TCATCTT             - #                  - #                      - #   17                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO: 10:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17 BASE - #PAIRS                                                  (B) TYPE:  NUCLEIC A - #CID                                                   (C) STRANDEDNESS:  SING - #LE                                                 (D) TOPOLOGY:  LINEAR                                                - -     (ii) MOLECULE TYPE:  OTHER NUCLEIC ACI - #D                           - -    (iii) HYPOTHETICAL:  NO                                                - -     (iv) ANTI-SENSE:  YES                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #10:                          - - TATCTGAGCA GCGCTCA             - #                  - #                      - #   17                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO: 11:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17 BASE - #PAIRS                                                  (B) TYPE:  NUCLEIC A - #CID                                                   (C) STRANDEDNESS:  SING - #LE                                                 (D) TOPOLOGY:  LINEAR                                                - -     (ii) MOLECULE TYPE:  OTHER NUCLEIC ACI - #D                           - -    (iii) HYPOTHETICAL:  NO                                                - -     (iv) ANTI-SENSE:  NO                                                  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #11:                          - - AGGCGCTGCC CCCACCA             - #                  - #                      - #   17                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO: 12:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17 BASE - #PAIRS                                                  (B) TYPE:  NUCLEIC A - #CID                                                   (C) STRANDEDNESS:  SING - #LE                                                 (D) TOPOLOGY:  LINEAR                                                - -     (ii) MOLECULE TYPE:  OTHER NUCLEIC ACI - #D                           - -    (iii) HYPOTHETICAL:  NO                                                - -     (iv) ANTI-SENSE:  YES                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #12:                          - - TTCCGTCCCA GTAGATT             - #                  - #                      - #   17                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO: 13:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17 BASE - #PAIRS                                                  (B) TYPE:  NUCLEIC A - #CID                                                   (C) STRANDEDNESS:  SING - #LE                                                 (D) TOPOLOGY:  LINEAR                                                - -     (ii) MOLECULE TYPE:  OTHER NUCLEIC ACI - #D                           - -    (iii) HYPOTHETICAL:  NO                                                - -     (iv) ANTI-SENSE:  NO                                                  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #13:                          - - TGGAAGACTC CAGTGGT             - #                  - #                      - #   17                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO: 14:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17 BASE - #PAIRS                                                  (B) TYPE:  NUCLEIC A - #CID                                                   (C) STRANDEDNESS:  SING - #LE                                                 (D) TOPOLOGY:  LINEAR                                                - -     (ii) MOLECULE TYPE:  OTHER NUCLEIC ACI - #D                           - -    (iii) HYPOTHETICAL:  NO                                                - -     (iv) ANTI-SENSE:  YES                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #14:                          - - CTTGAGTTCC AAGGCCT             - #                  - #                      - #   17                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO: 15:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17 BASE - #PAIRS                                                  (B) TYPE:  NUCLEIC A - #CID                                                   (C) STRANDEDNESS:  SING - #LE                                                 (D) TOPOLOGY:  LINEAR                                                - -     (ii) MOLECULE TYPE:  OTHER NUCLEIC ACI - #D                           - -    (iii) HYPOTHETICAL:  NO                                                - -     (iv) ANTI-SENSE:  NO                                                  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #15:                          - - CGCACCGAAT AGTTACG             - #                  - #                      - #   17                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO: 16:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17 BASE - #PAIRS                                                  (B) TYPE:  NUCLEIC A - #CID                                                   (C) STRANDEDNESS:  SING - #LE                                                 (D) TOPOLOGY:  LINEAR                                                - -     (ii) MOLECULE TYPE:  OTHER NUCLEIC ACI - #D                           - -    (iii) HYPOTHETICAL:  NO                                                - -     (iv) ANTI-SENSE:  YES                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #16:                          - - CCAGCGTGTC CAGGAAG             - #                  - #                      - #   17                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO: 17:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17 BASE - #PAIRS                                                  (B) TYPE:  NUCLEIC A - #CID                                                   (C) STRANDEDNESS:  SING - #LE                                                 (D) TOPOLOGY:  LINEAR                                                - -     (ii) MOLECULE TYPE:  OTHER NUCLEIC ACI - #D                           - -    (iii) HYPOTHETICAL:  NO                                                - -     (iv) ANTI-SENSE:  NO                                                  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #17:                          - - CTTCCTGGAC ACGCTGG             - #                  - #                      - #   17                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO: 18:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17 BASE - #PAIRS                                                  (B) TYPE:  NUCLEIC A - #CID                                                   (C) STRANDEDNESS:  SING - #LE                                                 (D) TOPOLOGY:  LINEAR                                                - -     (ii) MOLECULE TYPE:  OTHER NUCLEIC ACI - #D                           - -    (iii) HYPOTHETICAL:  NO                                                - -     (iv) ANTI-SENSE:  YES                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #18:                          - - CTGTAGGACC TTCGGTG             - #                  - #                      - #   17                                                                 __________________________________________________________________________

We claim:
 1. A method for early detecting an ovarian carcinoma in asubject comprising the steps of:taking a biological sample from saidsubject, the sample containing a p16 mRNA derived from said subject'sovarian epithelium; isolating the p16 mRNA from the sample; preparingcomplementary cDNA to the p16 mRNA; combining the prepared cDNA withprimers complementary to p16 DNA target sequences and to a control DNAtarget sequences; amplifying the DNA in the sample to produceamplification products; quantitating the amplification products; andcomparing the quantity of p16 target sequence amplification product inthe subject's sample against the quantity of p16 target sequenceamplification product from a similarly treated reference sample todetect a change in the subject's ovarian epithelium relative to thereference sample.
 2. The method of claim 1 wherein the reference sampleis taken from the group consisting of a sample from the same subject, anormal sample from a subject known to be ovarian tumor free, and asample from a subject known to have an ovarian tumor.
 3. The method ofclaim 1 where in the comparing step, the quantity of p16 target sequenceamplification product in the subject's sample is compared against acalibrated chart of p16 target sequence amplification product fromsimilarly treated normal samples to detect a change in the subject'sovarian epithelium relative to normal subjects.
 4. The method of claim 1for the early detection of an ovarian tumor in a subject where in thecomparing step, the quantity of p16 target sequence amplificationproduct in the subject's sample is compared against the quantity of p16target sequence amplification product from a similarly treated samplesfrom subjects known to have ovarian tumors.
 5. A method for producing acalibrated chart comprising the steps of:collecting a biological samplefrom each member of a first group of subjects known to bear an ovariantumor, and from each member of a second group of subjects known to befree of an ovarian tumor; and quantitating the presence of a p16 geneproduct in each sample, the gene product selected from the groupconsisting of p16 cDNA, p16 mRNA and p16 protein; and making a chartthat shows the quantity of the p16 gene product for the tumor-bearingversus the tumor-free groups of subjects.
 6. A method for earlydetection of ovarian tumors comprising the steps of:collecting a samplefrom a subject to be tested for the presence of an ovarian tumor; anddetermining p16 protein expression in the sample to early detect thepresence of an ovarian tumor.